Perelman School of Medicine at the University of Pennsylvania 22 articles published in JoVE Biology Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium Kole H. Buckley1, Keely A. Beyries1, Sandra Ryeom2, Sam S. Yoon2, Bryson W. Katona1 1Division of Gastroenterology and Hepatology, University of Pennsylvania Perelman School of Medicine, 2Department of Surgery, Columbia University Irving Medical Center Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy. Medicine Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor Rene Machietto1, Nicholas Giacobbe1, Jessica Perazzelli2, Ted J. Hofmann2, Allison Barz Leahy2,3, Stephan A. Grupp1,2,3, Yongping Wang1,3,4, Stephan Kadauke1,3,4 1Division of Oncology, Department of Pediatrics, Children’s Hospital of Philadelphia, 3Perelman School of Medicine at the University of Pennsylvania, 4 This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial. Neuroscience Maintenance of a Lateral Fluid Percussion Injury Device Anthony M. Farrugia2, Samuelle A. S. Delcy2,3, Brian N. Johnson1, Akiva S. Cohen1,2 1 Proper care and maintenance are essential for a lateral fluid percussion injury (LFPI) device to function reliably. Here, we demonstrate how to properly clean, fill, and assemble an LFPI device, and ensure that it is adequately maintained for optimal results. Behavior Comparative Analysis of Experimental Methods to Quantify Animal Activity in Caenorhabditis elegans Models of Mitochondrial Disease Manuela Lavorato*1, Neal D. Mathew*1, Nina Shah1, Eiko Nakamaru-Ogiso1,2, Marni J. Falk1,2 1 This study presents protocols for two semi-automated locomotor activity analysis approaches in C. elegans complex I disease gas-1(fc21) worms, namely, ZebraLab (a medium-throughput assay) and WormScan (a high-throughput assay) and provide comparative analysis among a wide array of research methods to quantify nematode behavior and integrated neuromuscular function. Neuroscience A Rapid Food-Preference Assay in Drosophila John O. Mack1, Yali V. Zhang1,2 1Monell Chemical Senses Center, 2Department of Physiology, The Diabetes Research Center, University of Pennsylvania Perelman School of Medicine We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies. Medicine Cooling or Warming the Esophagus to Reduce Esophageal Injury During Left Atrial Ablation in the Treatment of Atrial Fibrillation Jason Zagrodzky1, Mark M. Gallagher2, Lisa W. M. Leung2, Tiffany Sharkoski3, Pasquale Santangeli4, Cory Tschabrunn4, Jose M. Guerra5, Bieito Campos5, John MacGregor6, Jamal Hayat2, Brad Clark7, Alex Mazur8, Marcel Feher9, Martin Arnold9, Mark Metzl10, Jose Nazari10, Erik Kulstad11 1 The goal of this protocol is to describe the use of esophageal temperature modulation to counteract esophageal thermal injury from left atrial ablation for the treatment of atrial fibrillation. Cancer Research Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy Saba Ghassemi1, Michael C. Milone1 1Center for Cellular Immunotherapies, Perelman School of Medicine at the University of Pennsylvania We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo. Neuroscience Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo Joseph R. Patterson1, Nicole K. Polinski2, Megan F. Duffy1,3, Christopher J. Kemp1, Kelvin C. Luk4, Laura A. Volpicelli-Daley5, Nicholas M. Kanaan1, Caryl E. Sortwell1,3,6 1Department of Translational Science and Molecular Medicine, Michigan State University, 2 The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo. Biology Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit Jascha Brettschneider*1, Jason M. Correnti*1, Chelsea Lin1, Bianca Williams1, Amanke Oranu1, Amy Kuriakose1, Dru McIver-Jenkins1, Abigail Haba1, Isabelle Kaneza1, Sookyoung Jeon1, Eleonora Scorletti1, Rotonya M. Carr1 1Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit. Biochemistry A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells Xiongwei Cai1,2,3, Yi Zheng1, Nancy A. Speck2 1 A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer. Medicine Taste Exam: A Brief and Validated Test Jennifer E. Douglas1,2, Corrine J. Mansfield2, Charles J. Arayata2, Beverly J. Cowart2, Lauren R. Colquitt2, Ivy W. Maina1,2, Mariel T. Blasetti3, Noam A. Cohen3, Danielle R. Reed2 1Perelman School of Medicine at the University of Pennsylvania, 2Monell Chemical Senses Center, 3Department of Otorhinolaryngology-Head and Neck Surgery, Division of Rhinology, Hospital of the University of Pennsylvania This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject's reported sensation and taste receptor genotype. Immunology and Infection Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue Maria Steblyanko1, Nadia Anikeeva1, Marcus Buggert2,3, Michael R. Betts2, Yuri Sykulev4 1Department of Microbiology and Immunology, Thomas Jefferson University, 2Department of Microbiology and Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, 3Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital Huddinge, 4Departments of Microbiology and Immunology and Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood. Medicine Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy Xuehai Chen1, Rong Yu2, Ziqiang Xu3, Yan Zhang 3, Chengyang Liu4, Bicheng Chen*1, Hao Jin*3 1Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, 2Reproductive Center, The First Affiliated Hospital of Wenzhou Medical University, 3Department of Transplantation, The First Affiliated Hospital of Wenzhou Medical University, 4Department of Surgery, Perelman School of Medicine at the University of Pennsylvania Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava. Genetics Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome Vid Leko1,2, Smitha Sripathy1, Robin L. Adrianse1, Taylor Loe1, Angela Park1, Uyen Lao1, Eric J. Foss1, Marisa S. Bartolomei3, Antonio Bedalov1,4 1Clinical Research Division, Fred Hutchinson Cancer Research Center, 2Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 3Epigenetics Program, Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 4Departments of Medicine and Biochemistry, University of Washington We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome. Neuroscience Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors David J. Tischfield1,2, Stewart A. Anderson1 1Neuroscience Graduate Group, Perelman School of Medicine, University of Pennsylvania, 2Department of Psychiatry, Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications. Biochemistry Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions Robert Warneford-Thomson1,4, Chongsheng He1,2, Simone Sidoli1,3, Benjamin A. Garcia1,3, Roberto Bonasio1,2 1Epigenetics Institute, University of Pennsylvania Perelman School of Medicine, 2Department of Cell and Developmental Biology, University of Pennsylvania Perelman School of Medicine, 3Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, 4Graduate Group in Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry. Developmental Biology Stimulation of Notch Signaling in Mouse Osteoclast Precursors Gurpreet Kaur1,2, Jaimo Ahn1, Kurt D. Hankenson1,3, Jason W. Ashley1,4 1Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, 2Department of Biological Sciences, University of the Sciences, 3The Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, 4Department of Biology, College of Science, Technology, Engineering, & Mathematics, Eastern Washington University Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors. Cancer Research Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors Alan J. Fox1, Matthew C. Hiemenz1, David B. Lieberman1, Shrey Sukhadia1, Barnett Li1, Joseph Grubb1, Patrick Candrea1, Karthik Ganapathy1, Jianhua Zhao1, David Roth1, Evan Alley2,3, Alison Loren2,3, Jennifer J. D. Morrissette1 1Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, 2Division of Hematology/Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, 3Abramson Cancer Center This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information. Biology Assays for the Degradation of Misfolded Proteins in Cells Lili Guo1,2, Wil Prall1, Xiaolu Yang1 1Department of Cancer Biology, University of Pennsylvania Perelman School of Medicine, 2Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening. Biology Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models Meredith E. Jackrel1, Amber Tariq1, Keolamau Yee1, Rachel Weitzman1, James Shorter1 1Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast. Bioengineering The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits Joshua B. Slee1,2, Ivan S. Alferiev1,2, Robert J. Levy1,2, Stanley J. Stachelek1,2 1 Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. Biology Isolation of Neonatal Extrahepatic Cholangiocytes Sara Karjoo1, Rebecca G. Wells2 1 A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.
